JP2021038213A - Persea americana extract and preparation method thereof as well as uses thereof in anti-aging cosmetics - Google Patents

Abstract

To provide Persea americana extract, and to provide a preparation method thereof, as well as to provide uses thereof in anti-aging cosmetics.SOLUTION: Provided is a process of obtaining a Persea americana extract by treating the Persea americana through steps such as denuclearization, peeling, lump cutting, drying, crushing, sieving, ultrasonic assisted extraction, cooling, centrifugation, vacuum filtration, membrane ultrafiltration, extraction, and vacuum freeze-drying.EFFECT: The present invention can better prevent loss of the activity of nicotinamide mononucleotide that is an active ingredient in Persea americana pulp, and can also increase the yield of nicotinamide mononucleotide in the Persea americana extract. The Persea americana extract has an excellent anti-aging effect, effectively prevents rough skin and aging, reduces the appearance of wrinkles on the skin, and can keep the skin fresh and elastic.SELECTED DRAWING: Figure 1

Description

The present invention relates to the field of natural plant extraction technology, and particularly to avocado extract and its preparation method and its use in anti-aging cosmetics.

Nicotinamide mononucleotide (NMN) is a biochemical present in living cells, the product of the nicotinamide phosphoribosyl transferase reaction, and one of the important precursors of NAD +. Nicotinamide mononucleotide, as an intermediate in the repair pathway of NAD +, has antioxidant and oxidative stress reducing effects, has excellent antiaging and antioxidant effects, enhances energy metabolism, and physiologically declines the body. Can be delayed. Currently, it is mainly applied in the treatment of stroke, Alzheimer's disease, Parkinson's disease, retinal degenerative disease, and type 2 diabetes, not in the field of related cosmetic / skin care products. Nicotinamide mononucleotide is an endogenous substance of the human body, and because of its high safety and good thermal stability, nicotinamide mononucleotide has a wide range of applications as an active substance in the field of cosmetics / skin care products. There is a prospect.

Currently, nicotinamide mononucleotides are obtained by microbial fermentation, chemical synthesis, or in vitro enzyme catalyst preparation. The chemical synthesis process has drawbacks such as high cost, low yield, and difficulty in mass production. Although microbial fermentation and in vitro enzyme catalyst can avoid solvent residue, each process is complicated. There are drawbacks such as the fact that the enzyme catalyst is easily deactivated. Compared with the above three preparation methods, nicotinamide mononucleotides extracted from natural fruits and vegetables have advantages such as low cost, high yield, and high biological activity. Nicotinamide mononucleotides are widely present in natural foods such as avocado, broccoli, cabbage, edamame, cucumber, mushrooms, raw beef and shrimp. Since avocado is a nutritious fruit containing various vitamins, abundant fatty acids, trace elements, etc., it is possible to extract nicotinamide mononucleotide from avocado and apply it to the field of cosmetics or skin care product technology. It can solve the problem that the synthesis of nicotinamide mononucleotide in the technique is difficult. In general, different extraction processes will result in different final products extracted, and if the components of the extracted extract are too complex, they will cause poor symptoms such as skin allergies when applied to cosmetics or skin care products. May cause. Therefore, it is highly necessary to provide an efficient and hyposensitive antioxidant and anti-aging extract by improving the extraction process.

In order to overcome the above-mentioned problems existing in the prior art, an object of the present invention is to provide a method for preparing a high-yield, low-sensitization avocado extract so as to solve the above-mentioned drawbacks. ..

The present invention provides a method for preparing an avocado extract, the preparation method.
Avocado is removed, denuclearized and peeled, the pulp is cut into chunks, dried, crushed, sieved and then sieved to obtain avocado powder S1 and
The avocado powder according to step S1 is taken, water is added for ultrasonic auxiliary extraction, cooling is performed, centrifugation is performed, and the mixture obtained after centrifugation is subjected to vacuum filtration and membrane ultrafiltration, and the membrane ultrafiltration is performed. S2 for collecting the obtained filtrate and
The filtrate according to step S2 is extracted with ethyl acetate, the layers are separated, and then the lower layer liquid is taken from S3.
The underlayer liquid according to step S3 is vacuum freeze-dried to obtain a solid or a solid, which is included in S4.

Further, the number of meshes of the sieve in step S1 is 28 to 200 meshes.

Further, the ratio of the mass of the avocado powder to the volume of water is 1 to 25 g: 100 mL.

Further, the temperature of the ultrasonic auxiliary extraction is 45 to 80 ° C., the time of the ultrasonic auxiliary extraction is 30 to 120 min, and the power of the ultrasonic auxiliary extraction is 100 to 400 W.

Further, the pore size of the filter paper for vacuum filtration is 10 to 25 μm, and the molecular weight cut-off of the ultrafiltration membrane during the ultrafiltration is 1 KD to 5 KD.

Further comprising step S5 is to place the solid or solid frozen in a vacuum freeze-dryer in a nitrogen sealed drying box, dry for 30-60 minutes, and store in a sealed manner.

Further, under the vacuum freeze-drying conditions of step S4, the vacuum pressure is 50 to 150 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, and the temperature in the first stage is set to 355 to -25 ° C. , Hold for 1-2 hours, set the temperature of the second stage to -20 to -10 ° C, hold for 0.5 to 2 hours, set the temperature of the third stage to -10 to -5 ° C. Hold for 3 to 5 hours, set the temperature of the analysis drying stage to 30 to 60 ° C., and hold for 7 to 12 hours.

Furthermore, the present invention further provides an avocado extract prepared according to the above preparation method.

Furthermore, the present invention further provides the use of the avocado extract prepared by the above preparation method in anti-aging cosmetological and skin care products, wherein the dose of the avocado extract is 0.05 to 99% by weight. is there.

Further, the anti-aging beauty / skin care product is one or more of lotions, essences, undiluted solutions, emulsions, creams, masks, tablets and rouge.

Avocado has an extremely complex chemical composition as a natural food. Applicants said that in the course of the experiment, the ultrasonically assisted avocado extract contained a small amount of γ-sitosterol, and γ-sitosterol had the effect of increasing allergic reactions such as allergic contact dermatitis of the skin. I found that there is.

Therefore, the applicant extracts the filtrate obtained by ultrasonic auxiliary extraction in the extraction process with ethyl acetate, because nicotine amide mononucleotide is easily dissolved in water and γ-sitosterol is poorly soluble in water. By removing a small amount of γ-sitosterol in the filtrate by operation, the subsequent vacuum freeze-dried avocado extract is γ-sitosterol-free and is safe as it applies to the cosmetic or skin care products of the avocado extract. Guaranteed.

Next, the applicant also found in the course of the experiment that the content of nicotinamide mononucleotide in the avocado extract obtained by temperature gradient controlled drying during the sublimation drying step of vacuum drying was higher, which is the temperature. This is because the gradient control does not convert the relevant chemical components in the filtrate due to sudden changes in the environment. In addition, by placing the avocado extract in a nitrogen-sealed drying box after vacuum drying and drying it for a certain period of time, the stability of the nicotinamide mononucleotide in the extract can be more reliably ensured, which is unexpected for the applicant. The cause is that the surface area of the product after vacuum drying increases, moisture and microorganisms are easily adsorbed, and it may lead to conversion and change of chemical components.

Therefore, the preparation method of the present invention has the following beneficial effects as compared with the conventional technique.

(1) The present invention uses an ultrasonic auxiliary water extraction method to extract a nicotine amide monosolvent from avocado, which is organic in a short time and at low cost while realizing large-scale factory production. There is no residual solvent, water is used as the solvent, it is highly safe, the lower layer products are treated using vacuum freeze-drying in the preparation method, and three-step temperature gradient controlled drying is performed in the sublimation drying step of vacuum drying. Thereby, the loss of the activity of the nicotine amide mononucleotide which is an active ingredient in the avocado fruit meat can be better prevented, and the yield of the nicotine amide mononucleotide in the avocado extract can be improved.

(2) The present invention applies an avocado extract to anti-aging cosmetics for the first time, and in addition to nicotinamide mononucleotide, the extract contains various active ingredients such as flavones, polysaccharides, and polypeptides, and these active ingredients. Has an excellent anti-aging effect, can effectively prevent rough skin and aging, reduce the appearance of wrinkles on the skin, and keep the skin fresh and elastic.

(3) In the method for preparing the avocado extract of the present invention, the filtrate obtained after the ultrasonic auxiliary extraction is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer clear liquid is taken and vacuum dried. Improves the purity of nicotine amide mononucleotide in the avocado extract, removes γ-citosterol contained in the avocado extract, and subsequently improves biosafety in the process of the extract being used in cosmetics, allergies The occurrence of the reaction can be significantly reduced.

(4) In the method for producing an avocado extract of the present invention, the avocado extract frozen in a vacuum freeze-dryer is placed in a nitrogen-sealed drying box, dried for a certain period of time, and then sealed and stored to produce nicotine in the extract. It stores amide mononucleotides for a longer period of time, better guarantees that the activity stabilizes within a certain period of time, increases the surface area of the avocado extract due to low temperature freeze-drying, increases the hygroscopicity, and the activity of the extract is poor. It is stable and can be prevented from being prone to oxidative rot.

FIG. 1 is a calibration curve of the nicotinamide mononucleotide according to the present invention.

Hereinafter, the present invention will be further described through description of specific embodiments, but this does not limit the present invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the present invention. All are within the scope of the present invention, as long as they do not deviate from the basic idea of the present invention.

Example 1
The method for preparing the avocado extract of Example 1 is as follows.

For S1, remove the avocado, denuclearize and peel, cut the flesh into small pieces of 1 cm x 1 cm, dry at 45 ° C. to a certain weight, grind, and sieve through a 28-mesh sieve. Is.

In S2, 2.5 kg of the avocado powder described in step S1 is precisely weighed, 10 L of water is added, ultrasonic auxiliary extraction is performed under the conditions of a temperature of 45 ° C. and an ultrasonic power of 100 W for 120 minutes, and the mixture is left at room temperature for natural cooling. After that, the mixture is centrifuged at a rotation speed of 4500 mp for 20 minutes, filtered under reduced pressure with a filter paper having a pore size of 10 μm, and then ultrafiltered with a filtration membrane having a fractional molecular weight of 1 KD to collect the filtrate.

In S3, the filtrate according to step S2 is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer liquid is taken.

In S4, the lower layer liquid is vacuum freeze-dried, the vacuum pressure is set to 150 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to −35 ° C., and the temperature is maintained for 1 hour. Then, set the temperature of the second stage to -20 ° C and hold for 2 hours, set the temperature of the third stage to -10 ° C, hold for 3 hours, and set the temperature of the analysis drying stage to 30 ° C. And keep at constant temperature for 12 hours to obtain a solid or solid, i.e. avocado extract.

Example 2
The method for preparing the avocado extract of Example 2 is as follows.

For S1, avocado is removed, denuclearized and peeled, the flesh is cut into small lumps of 0.5 cm × 0.5 cm, dried at 60 ° C. to a certain weight, crushed, and sieved through a 200 mesh sieve. It is to sift.

In S2, 0.1 kg of the avocado powder described in step S1 is precisely weighed, 10 L of water is added, ultrasonic auxiliary extraction is performed for 30 minutes under the conditions of a temperature of 80 ° C. and an ultrasonic power of 400 W, and the mixture is left at room temperature for natural cooling. After that, the mixture is centrifuged at a rotation speed of 4500 mp for 20 minutes, filtered under reduced pressure with a filter paper having a pore size of 25 μm, and then ultrafiltered with a filtration membrane having a fractional molecular weight of 5 KD to collect the filtrate.

In S3, the filtrate according to step S2 is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer liquid is taken.

In S4, the lower layer liquid is vacuum freeze-dried, the vacuum pressure is set to 50 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to -25 ° C, and the temperature is maintained for 2 hours. Then, the temperature of the second stage is set to -10 ° C and held for 0.5 hours, the temperature of the third stage is set to -5 ° C and held for 5 hours, and the temperature of the analysis drying stage is set to 60 ° C. To obtain a solid or solid, i.e. avocado extract, set to constant temperature for 7 hours.

Example 3
The method for preparing the avocado extract of Example 3 is as follows.

For S1, avocado is removed, denuclearized and peeled, the flesh is cut into 1 cm × 1 cm lumps, dried at 45 ° C. to a constant weight, crushed, and sieved with a 28-mesh sieve. To obtain an avocado powder having a particle size of less than 250 μm.

In S2, 2.5 kg of the avocado powder described in step S1 is precisely weighed, 10 L of water is added, ultrasonic auxiliary extraction is performed under the conditions of a temperature of 45 ° C. and an ultrasonic power of 100 W for 120 minutes, and the mixture is left at room temperature for natural cooling. After that, the mixture is centrifuged at a rotation speed of 4500 mp for 20 minutes, filtered under reduced pressure with a filter paper having a pore size of 10 μm, and then ultrafiltered with a filtration membrane having a fractional molecular weight of 1 KD to collect the filtrate.

In S3, the filtrate according to step S2 is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer liquid is taken.

In S4, the lower layer liquid is vacuum freeze-dried, the vacuum pressure is set to 150 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to −35 ° C., and the temperature is maintained for 1 hour. Then, set the temperature of the second stage to -20 ° C and hold for 2 hours, set the temperature of the third stage to -10 ° C, hold for 3 hours, and set the temperature of the analysis drying stage to 30 ° C. And keep at constant temperature for 12 hours to obtain a solid or solid.

S5 is to put the vacuum freeze-dried solid or solid in a nitrogen-sealed drying box, dry it for 30 minutes, and store it in a sealed manner to obtain an avocado extract.

Example 4
The method for preparing the avocado extract of Example 4 is as follows.

For S1, avocado is removed, denuclearized and peeled, the flesh is cut into small lumps of 0.5 cm × 0.5 cm, dried at 60 ° C. to a certain weight, crushed, and sieved through a 200 mesh sieve. It is to sift.

In S2, 0.1 kg of the avocado powder described in step S1 is precisely weighed, 10 L of water is added, ultrasonic auxiliary extraction is performed for 30 minutes under the conditions of a temperature of 80 ° C. and an ultrasonic power of 400 W, and the mixture is left at room temperature for natural cooling. After that, the mixture is centrifuged at a rotation speed of 4500 mp for 20 minutes, filtered under reduced pressure with a filter paper having a pore size of 25 μm, and then ultrafiltered with a filtration membrane having a fractional molecular weight of 5 KD to collect the filtrate.

In S3, the filtrate according to step S2 is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer liquid is taken.

In S4, the lower layer liquid is vacuum freeze-dried, the vacuum pressure is set to 50 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to -25 ° C, and the temperature is maintained for 2 hours. Then, the temperature of the second stage is set to -10 ° C and held for 0.5 hours, the temperature of the third stage is set to -5 ° C and held for 5 hours, and the temperature of the analysis drying stage is set to 60 ° C. And keep at constant temperature for 7 hours to obtain a solid or solid.

S5 is to put the vacuum freeze-dried solid or solid in a nitrogen-sealed drying box, dry it for 60 minutes, and store it in a sealed manner to obtain an avocado extract.

Example 5
The method for preparing the avocado extract of Example 5 is as follows.

For S1, avocado is removed, denuclearized and peeled, the flesh is cut into small lumps of 0.5 cm × 0.5 cm, dried at 50 ° C. to a certain weight, crushed, and sieved through a 100 mesh sieve. Sifting to obtain avocado powder with a particle size of less than 400 μm.

In S2, 1 kg of the avocado powder described in step S1 was precisely weighed, 10 L of water was added, ultrasonic auxiliary extraction was performed under the conditions of a temperature of 55 ° C. and an ultrasonic power of 200 W for 60 minutes, and the mixture was allowed to stand at room temperature for natural cooling. After that, the mixture is centrifuged at a rotation speed of 4500 mp for 20 minutes, filtered under reduced pressure with a filter paper having a pore size of 20 μm, and then ultrafiltered with a filtration membrane having a fractional molecular weight of 3 KD to collect the filtrate.

In S3, the filtrate according to step S2 is extracted with ethyl acetate, allowed to stand for layer separation, and then the lower layer liquid is taken.

In S4, the lower layer liquid is vacuum freeze-dried, the vacuum pressure is set to 100 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to −30 ° C., and 1.5. Hold for hours, set the temperature of the second stage to -15 ° C, hold for 1 hour, set the temperature of the third stage to -8 ° C, hold for 3 hours, and set the temperature of the analysis drying stage to 45 ° C. And keep at a constant temperature for 10 hours to obtain a solid or solid.

S5 is to put the vacuum freeze-dried solid or solid in a nitrogen-sealed drying box, dry it for 45 minutes, and store it in a sealed manner to obtain an avocado extract.

Comparative Example 1
Compared to Example 5, the ultrasonic auxiliary extraction temperature in step S2 is set to 90 ° C., and the other steps and their operating conditions are consistent with Example 5.

Comparative Example 2
Compared to Example 5, the sublimation drying step in the vacuum freeze-drying step S4 does not use the three-step temperature gradient controlled drying, the temperature is directly set to −8 ° C. and held for 5.5 hours, and the other steps. And its operating conditions are consistent with Example 5.

Comparative Example 3
Compared to Example 5, the extraction operation with ethyl acetate was not performed, the filtrate filtered in step S2 was directly vacuum freeze-dried, and the other steps and the operating conditions were the same as in Example 5.

Test Example 1, Measurement of nicotinamide mononucleotide and γ-sitosterol content by high performance liquid chromatography The avocado extracts prepared in Examples 1 to 5 and Comparative Examples 1 to 3 were taken and tested.

1.1 Measurement of nicotinamide mononucleotide content 1.1.1, sample pretreatment Approximately 0.2 g of each of the test samples of the avocado extracts of Examples 1 to 5 and Comparative Examples 1 to 3 is accurately measured up to 1 mg. Weighed in, 10 ml of pure water was added to dissolve the mixture, and the mixture was ultrasonically treated with an ultrasonic cleaner for 20 minutes, then scalloped to 20 mL with pure water and mixed uniformly. A part of the solution is injected into a centrifuge tube, centrifuged at 4500 r / min for 30 minutes, the supernatant is taken, and the supernatant is filtered through a 0.45 μm filtration membrane to prepare the test. Two parallel tests were performed, and the measurement results of the two parallel tests were required to have a relative deviation of 5% or less.

11.2, Chromatographic Conditions Chromatographic Column: Agilent XDB C18 Column (250 mm x 4.6 mm, 5 μm); Flow Rate: 1.0 mL / min;
Sample injection volume: 20 μL; column temperature: 25 ° C.; measurement wavelength: 260 nm.
Mobile phase: Acetonitrile-pH 6 phosphate buffer, gradient elution conditions (volume ratio) are as follows.

1.1.3, Preparation of nicotinamide mononucleotide calibration curve Prepare a stock solution of 1 mg / mL nicotinamide mononucleotide with pure water, and prepare 5 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / Accurately dilute mL of the solution to be measured with pure water and inject into liquid chromatography according to the chromatography conditions of 1.1.2, and from the chromatogram, the retention time of nicotinamide mononucleotide is 2.539 min. Obtained, a calibration curve of nicotinamide mononucleotide was prepared with the concentration of nicotinamide mononucleotide as the horizontal axis and the peak area as the vertical axis, and is shown in FIG.

1.1.4, Sample Test A test sample of avocado extract was injected into liquid chromatography according to the chromatography conditions of 1.1.2, and two parallel samples of each Example / Comparative Example at 2.539 min. The peak area of the chromatography peak was recorded, the content of nicotinamide mononucleotide in the test sample of avocado was calculated from the calibration curve of nicotinamide mononucleotide, and the average value of the two test results was taken as the final value. Table 2 shows the content of nicotinamide mononucleotide in each sample.

1.2 Measurement of γ-sitosterol content 1.2.1, sample pretreatment 0.2 g of each of the test samples of the avocado extract of Example 5 and Comparative Example 3 was accurately weighed to 1 mg, and 10 ml. 95% ethanol was added to dissolve the mixture, and the mixture was ultrasonically treated with an ultrasonic cleaner for 20 minutes, cooled to room temperature, and then squeezed up to 20 mL with 95% ethanol and mixed uniformly. A part of the solution is injected into a centrifuge tube, centrifuged at 4500 r / min for 30 minutes, the supernatant is taken, and the supernatant is filtered through a 0.45 μm filtration membrane to prepare the test. Two parallel tests were performed, and the measurement results of the two parallel tests were required to have a relative deviation of 5% or less.

1.2.2, Chromatographic Conditions Chromatographic Column: Agilent XDB C18 Column (250 mm x 4.6 mm, 5 μm); Flow Rate: 1.0 mL / min;
Sample injection volume: 20 μL; column temperature: 30 ° C.; measurement wavelength: 210 nm.
Mobile phase: acetonitrile-isopropanol, volume ratio 75:25; isocratic elution was performed.

1.2.3, Preparation of γ-citosterol calibration curve Prepare a 1 mg / mL stock solution of γ-citosterol in pure water and prepare 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL. The solution to be measured is accurately diluted with 95% ethanol and injected into liquid chromatography according to the chromatography conditions of 1.2.2. γ-Citosterol with the concentration of γ-citosterol on the horizontal axis and the peak area on the vertical axis. However, the holding time of γ-citosterol is 16.157 min.

1.2.4, Sample Test The test sample of 1.2.1 is injected into liquid chromatography according to the chromatography conditions of 1.2.2 and two parallels of each Example / Comparative Example at 16.157 min. Record the peak area of the chromatographic peak of the sample, calculate the content of γ-sitosterol in the test sample of avocado from the calibration curve of γ-sitosterol, and take the average value of the two test results to make the final value. The test result was good.

As can be seen from Table 2, the content of nicotinamide mononucleotide in the avocado extract obtained by the preparation method of the present invention is 8.026 to 8.828 μg / mg, of which Example 5 is the present invention. This is an optimal example.

In Comparative Example 1, the ultrasonic auxiliary extraction temperature in step S2 was set to 90 ° C., and the other steps were consistent with Example 5, but the content of nicotinamide mononucleotide in the avocado extract was significantly reduced. Compared to Example 5, it was clear that approximately 27.8% was lost, indicating that the extraction temperature had a clear effect on the content of nicotinamide mononucleotides in the avocado extract.

In Comparative Example 2, the temperature was directly set to -8 ° C. and held for 5.5 hours in the sublimation drying step of vacuum freeze-drying in step S4 without using the three-step temperature gradient control drying, and compared with Example 5. Then, the content of nicotinamide mononucleotide in the avocado extract is also reduced, that is, the three-step temperature gradient controlled drying operation is prevented from being affected by the biological activity of the nicotinamide mononucleotide in the avocado extract. Further, the yield of the product in the extraction process can be improved.

Comparative Example 3 shows that the extraction operation does not reduce the content of the nicotinamide mononucleotide because the content of the nicotinamide mononucleotide is close to that of Example 5, and it is tested by high performance liquid chromatography in Example 5. And the content of γ-sitosterol in Comparative Example 3 was obtained to be 7.9 ug / mg and 0.84 ug / mg, respectively, and by extracting the filtrate, the content of γ-sitosterol was reduced, and cosmetics or cosmetics or Avoid possible allergic symptoms due to application in skin care products.

Test Example 2, Antioxidant and Anti-aging Performance Test 2.1, DPPH Elimination Experiment Test DPPH, also called 1,1-diphenyl-2-picrylhydrazil, is a very stable free radical centered on nitrogen. The stability is that the steric hindrance of the three benzene rings, which mainly has a resonance stabilizing effect, is that the unpaired electrons on the nitrogen atom sandwiched between them play their own role in electron pair formation. Due to the inability to do so. The absolute ethanol solution is purple, has a maximum absorption wavelength at a wavelength of 517 nm, and has a linear relationship between absorbance and concentration. Adding a radical scavenger into it can reduce the number of radicals, reduce the absorbance and lighten the color of the solution in combination with or instead of DPPH, thereby assessing the ability to scavenging radicals. it can.

A 0.2 mmol / L DPPH absolute ethanol solution was prepared, a sample solution to be measured at a concentration of 200 mg / mL was prepared, and vitamin C was used as a positive control sample.

Aspirate 2 mL of the sample solution and 2 mL of the DPPH solution into a stoppered test tube, mix them uniformly, react for 30 minutes while avoiding light, measure the absorption value A1 at the 517 nm wavelength, and add 2 mL of the sample solution and 2 mL of methanol, respectively. Suction into a stoppered test tube to mix uniformly, avoid light and react for 30 minutes, measure the absorption value A2 at 517 nm wavelength, and suck 2 mL of DPPH solution and 2 mL of methanol into the stoppered test tube to make them uniform. And react for 30 minutes, avoiding light, measuring the absorption value A0 at 517 nm wavelength, each sample making 3 parallel samples, recording the DPPH elimination rate of the avocado extract, the final result is The average value of three parallel samples was taken.

2.2, Advanced glycation end product removal experimental test In non-enzymatic glycosylation, proteins and glucose undergo a non-enzymatic reaction in the body to form early glycosylation products such as Schiff base or Amadori products, which are further oxidized and re-glycosylated. It is a series of complex non-enzymatic reactions that form irreversible advanced glycation end products (AGEs) through processes such as sequencing and cross-linking. The reaction is wide and slow in vivo, causing protein dysfunction and aging, and can also cause aging and lesions of organic tissues, so substances that can efficiently remove AGEs have anti-aging effects. Can be presented to have.

The following reagents were prepared respectively. (1) 0.1 mol / L NaOH solution; (2) 0.05 mol / L PBS phosphate buffer (pH = 7.4); (3) 10 mg / mL bovine serum albumin solution; (4) 45 mg / mL glucose solution; (5) 0.25 mmol / L aminoguanidine hydrochloride solution; (6) 200 mg / mL avocado extract sample solution to be measured.
Place 0.05 mol / L PBS phosphate buffer (pH = 7.4), 10 mg / mL bovine serum albumin solution and 45 mg / mL glucose solution in a constant temperature and humidity incubator, and then measure the avocado extract. A sample solution and a 0.25 mmol / L aminoguanidine hydrochloride solution were added, and the mixture was cultured at 60 ° C. for 40 hours (corresponding to culturing at 30 ° C. for 60 days). After 40 hours, each sample was taken out, and after 40 hours, each sample was taken out and detected with a microplate reader, and the fluorescence absorption value at the fluorescence excitation / emission wavelength of 370/440 nm was recorded. For each sample, three parallel samples were prepared, the suppression rate of AEGs in the avocado extract was recorded, and the final result was the average value of the three parallel samples. Inhibition rate AEGs are shown in the following equation, provided that, A _sample is fluorescent when represents fluorescence intensity in the case of adding the test substance and glucose solution, A _{sample blank,} the addition of the test substance without addition of glucose solution The intensity was shown, the A- _{negative control} showed the fluorescence intensity in the presence of the glucose solution without the subject, and the A _{glucose-deficient negative control} showed the fluorescence intensity in the absence of the subject and the glucose solution.

As shown in Examples 1 to 5 of Table 3, the DPPH elimination rate and the AEGs suppression rate of the avocado extract prepared by the preparation method of the present invention are 86.58% to 88.99% and 85.38%, respectively. It is in the range of 89.83%, whereas the DPPH elimination rate and AEGs suppression rate of Comparative Example 1 are 71.26% and 68.79%, respectively, so that the DPPH elimination rate and the AEGs suppression rate are combined. It was shown that there was a positive correlation tendency with the content of nicotinamide mononucleotide. The test data in Table 2 showed that the avocado extract prepared by the preparation method of the present invention had excellent antioxidant and anti-aging effects.

Test Example 3, Stability Test of Avocado Extract Obtained by Different Preparation Methods Test Method: The avocado extract obtained by the preparation methods of Examples 1, 2, 5 and Comparative Example 3 was taken and tested. After the test samples were sealed and stored for 14, 28 and 56 days, respectively, the content of nicotinamide mononucleotide in the extract was tested by liquid chromatography, and the chromatography conditions and test method were consistent with Test Example 1. Specific test results are shown in Table 4.

As shown in Table 4, the nicotinamide mononucleotide contents of the avocado extracts of Examples 1 and 2 decreased after 14 to 56 days, and the nicotinamide mononucleotide content increased as the storage time increased. The content was further reduced, whereas the avocado extracts of Example 5 and Comparative Example 3 did not change much in the nicotinamide mononucleotide content after 14-56 days, and thus were frozen in a vacuum freeze dryer. It was demonstrated that the stability of the nicotinamide mononucleotide in the avocado extract can be improved by placing the dried product in a nitrogen-sealed drying box, drying it, and storing it in a sealed manner.

Those skilled in the art should understand that the above examples merely illustrate the principles of the present invention and its effects, and do not limit the present invention. Those skilled in the art may modify or modify the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or modifications made by one of ordinary skill in the art, without departing from the spiritual and technical ideas disclosed in the present invention, should still be included in the claims of the present invention.

Comparative Example 3 shows that the extraction operation does not reduce the content of the nicotinamide mononucleotide because the content of the nicotinamide mononucleotide is close to that of Example 5, and it is tested by high performance liquid chromatography in Example 5. And the content of γ-sitosterol in Comparative Example 3 was obtained to be 7.9 μg / mg and 0.84 μg / mg, respectively, and the content of γ-sitosterol was reduced by extracting the filtrate. And avoid possible allergic symptoms due to application in cosmetics or skin care products.

Claims (10)

Avocado is removed, denuclearized and peeled, the pulp is cut into chunks, dried, crushed, sieved and then sieved to obtain avocado powder S1 and
The avocado powder according to step S1 is taken, water is added for ultrasonic auxiliary extraction, cooling is performed, centrifugation is performed, and the mixture obtained after centrifugation is subjected to vacuum filtration and membrane ultrafiltration, and the membrane ultrafiltration is performed. S2 for collecting the obtained filtrate and
The filtrate according to step S2 is extracted with ethyl acetate, the layers are separated, and then the lower layer liquid is taken from S3.
A method for preparing an avocado extract, which comprises S4 in which the lower layer liquid according to step S3 is vacuum freeze-dried to obtain a solid or a solid. The method for preparing an avocado extract according to claim 1, wherein the number of meshes of the sieve in step S1 is 28 to 200 meshes. The method for preparing an avocado extract according to claim 1, wherein the ratio of the mass of the avocado powder to the volume of water is 1 to 25 g: 100 mL. The claim is characterized in that the temperature of the ultrasonic auxiliary extraction is 45 to 80 ° C., the time of the ultrasonic auxiliary extraction is 30 to 120 min, and the power of the ultrasonic auxiliary extraction is 100 to 400 W. The method for preparing an avocado extract according to 1. The avocado extract according to claim 1, wherein the filter paper for vacuum filtration has a pore size of 10 to 25 μm, and the fractional molecular weight of the ultrafiltration membrane during ultrafiltration is 1 KD to 5 KD. Preparation method. The avocado extract according to claim 1, further comprising step S5, wherein the solid or solid frozen in a vacuum freeze-dryer is placed in a nitrogen-sealed drying box, dried for 30 to 60 minutes, and stored in a sealed manner. Preparation method. The vacuum freeze-drying conditions in step S4 are that the vacuum pressure is 50 to 150 Pa, the temperature gradient is controlled in three stages in the sublimation drying stage, the temperature in the first stage is set to 35 to -25 ° C, and 1 to 1 Hold for 2 hours, set the temperature of the second stage to -20 to -10 ° C, hold for 0.5 to 2 hours, set the temperature of the third stage to -10 to -5 ° C, 3 to The method for preparing an avocado extract according to claim 1, wherein the avocado extract is held for 5 hours, the temperature of the analysis drying step is set to 30 to 60 ° C., and the temperature is held for 7 to 12 hours. An avocado extract prepared by the method for preparing an avocado extract according to any one of claims 1 to 7. The use of the avocado extract according to claim 8 in an anti-aging beauty / skin care product, wherein the dose of the avocado extract is 0.05 to 99% by weight. The avocado extract according to claim 8, wherein the anti-aging beauty / skin care product is one or more of lotions, essences, undiluted solutions, emulsions, creams, masks, tablets, and rouge. Use in anti-aging beauty and skin care products.